SABRe CVD Initiative noteworthy accomplishments in FY10: Project 1: Discovery Proteomics/Metabolomics 1. The completion of a QC pilot study The combined results of the proteomics/metabolomics QC pilot study were excellent. The FHS samples tested (N=70) have not suffered any significant degradation during their storage both in terms of protein and metabolite abundance or modifications/degradation of the detected proteins. These results exceed expectations. 2. Design completion of 3 sub-studies Project 1 uses analytical techniques that are not considered high throughput and are expensive, necessitating 3 sub-studies of about 600 samples each to focus the analytical measurement around a set of primary traits. There are 3 sub studies: New-Onset CVD Events, Coronary Artery Calcium (CAC), and Metabolic Risk Factors. 3. Development of analytical pipeline Study statisticians have been working with pilot study data to develop an analytical pipeline to streamline the QC and analysis of the large amount of data to be generated. They have created an interface that can report back some understandable QC plots reflecting the quality and distribution of the data. 4. Launch of proteomics for CVD cases and controls In April of 2010 the discovery proteomics analytical data collection commenced. The QC metrics are exceedingly high with >900 proteins meeting the defined QC criteria. Proteomic profiling of 136 MI cases and controls samples is anticipated to be completed in Oct-Nov 2010. Summary: Progress has been slow and deliberate for Project 1. Currently we are behind schedule, but feel the next year will be pivotal to catching up with the projected timelines for project completion. Project 2: Measure 180 circulating plasma proteins in >7000 FHS participant samples over 5 years. 1. Creation of a Biomarker Nomination Committee (BNC) and a nomination and review process The BNC was formed to handle the identification of proteins of interest to Project 2, creating a transparent review process and then executing these tasks. The committee has met bi-weekly and made excellent progress. A formal Biomarker Nomination process was developed and implemented. A template was created with the required elements needed for a thorough review of each nominated protein, including the following critical fields: a) Source of existing data on proposed target, b) The diseases associated with the marker;c) Is the marker pathway dependent? d) Is the marker Plieotropic? e) Have other large scale studies been conducted on this target? f) Relevant literature linked to marker 2. Literature mining of proteins to initiate the nomination process In collaboration with NCBI, mining of GWAS databases and aligning results with GEO databases for evidence of genomic convergence are underway (i.e. gene expression and GWAS signals pointing to a single gene for which a gene product can be targeted for assay). The current set of 31 nominated targets includes 30 biomarkers identified by conventional literature and bibliographic mining and review. There is one member of the current panel that has been identified through genomic convergence strategies. 3. The 1st panel of biomarkers for assay development and measurement After only six months of work on Project 2, the BNC has reviewed >60 targets of interest and has nominated 31 biomarkers for assay development and measurement by Si-Al. Summary: Project 2 is running well. The communications with Si-Al are going well and all parties are pleased with the progress being made on the set of initial target biomarkers, of which 31 out of 45 have been chosen. Project 3: RNA expression analysis 1. Completion of the Pilot Study The QC pilot study was a critical step for Project 3 since it was during this process that the selection of the RNA source material for the entire study was chosen. There were 4 RNA sources available: Paxgene (whole blood RNA), buffy coat samples, immortalized cell lines, and peripheral blood mononuclear cells (PBMCs). QC analyses were conducted looking at RNA yield and quality and quantity, genes expressed within from each RNA source, and identification of Y-chromosome transcripts to differentiate male vs. female samples. After looking at each QC element, it was decided to move forward with the Paxgene tube as the source of RNA. 2. Design of the CVD case control study To accelerate research opportunities for gene expression experiments, we front-loaded a CVD case-control study at the beginning of the laboratory work. In doing this, we anticipated that this effort would lead to early discoveries and publications. The incident CVD study includes 222 matched case-control pairs. 3. Gene expression analysis in the CVD study Five batches representing the 222 pairs in the CVD case-control study were processed in the lab and a complete gene expression database was generated. We are proceeding with analyses of differential expression at the gene and exon level. Analyses will be conducted on 287,329 exons and 22,011 genes. 4. RNA expression profiling of the FHS Offspring Cohort The laboratory is collecting expression data on the remaining 2000 samples from the FHS Offspring cohort. If the proposed timeline is maintained, completion of chipping of the FHS Offspring gene expression samples will be in late 2010 or early 2011. Summary: Gene expression profiling for Project 3 is proceeding. The lab has the ability to conduct relatively high throughput profiling while maintaining QC standards. The lab is processing samples and RNA isolation is currently ahead of schedule. Project 4: miRNA expression in >7000 FHS samples The project had a slow start in FY10 due to QC problems that necessitated an overhaul of miRNA profiling methods. A second QC study was completed, using the new methods, and the data were excellent and exceeded expectations. The data from QC study Panel A study were good. The mean %CV is very low (3.5%) and only a few points are found above the 10% CV line. As noted for Project 3, the same RNA samples are being utilized for the miRNA project, so the entire 2608 FHS Offspring cohort sample set is ready for the miRNA analysis to begin. It is anticipated that the Offspring samples will be profiled for up to 750 miRNA by the early 2011.